The gastric H,K-ATPase provides the enzymatic basis for acid secretion in the stomach. For the pathology leading to gastric ulcer diseases, the most efficacious treatment is the suppression of stomach acid with concomitant H pylori eradication. Though gastric and duodenal ulcers have historically been treated with H-2 blockers, future pylori eradication. Though gastric and duodenal ulcers have historically been treated with H-2 blockers, future alternatives such as that provided by the acid-activated thiol reagent, Omeprazole, may increasingly rely on the direct inhibition or modification of the H,K-ATPase. Reversible inhibitors, K plus competitive ligands of the H, K-ATPase, appear to interact at or near the cation binding domain. MDPQ, a novel fluorescent probe from this reversible class of pump inhibitors has been used to investigate the H,K-ATPase function at an inhibitor domain present in both the intact ATPase and a membrane preparation obtained by tryptic digestion of the enzyme. Insights gained of the cation binding domain in these preparations may aid in the design of future pharmacology, exploiting key features of pump function and structure. This work helps define an area of potential clinical relevance aimed at establishing the H, K-ATPase as a therapeutic target in ulcer disease. Progress will depend on the advance of knowledge of the H,K-ATPase structure and its relationship to function. A fundamental issue of this proposal is the elucidation of the structural basis of cation selectivity in Ion pumps. This focus is appropriate because the P type ATPase pumps have discriminate specificity and stoichiometry of cation transport whose structural basis is not evident from inspections of the 1 degree structure or comparisons of sequence homology between these pumps. The proposal provides a strategy to identify domains and residues within those domains that contribute to cation binding and ion translocation. It utilizes complementary strategies involving biochemical and molecular biological methodologies where residues within those domains that contribute to cation binding and ion translocation. It utilizes complementary strategies involving biochemical and molecular biological methodologies where residues implicated in studies of the native H, K-ATPase will provide a rational basis for refined structure-function studies utilizing site specific mutations. Together these studies will identify structural features of domains within the alpha and beta-subunits of the H,K-ATPase important for cation binding and will define residues essential for cation binding. This proposal focuses on the identification of charged residues within the transmembrane domain that are necessary for cation binding and the elucidation of the contribution of the beta-subunit to cation binding.